Targeting parasite-host communication to combat liver fluke-induced bile duct cancer PROJECT SUMMARY Liver fluke infection with Opisthorchis viverrini remains problematic in East Asia and is endemic in Thailand and Laos, where ~10 million people are infected. The public health implications of this situation are substantial since there is no stronger link between a human malignancy and a eukaryotic pathogen than that between cholangiocarcinoma (CCA) (bile duct cancer) and infection with the liver fluke O. viverrini. Northeast Thailand reports the highest incidence of CCA worldwide, with the 2014 CCA age standardized incidence rate (ASR) of 85 per 100,000, which equates to 26, 000 CCA-related deaths annually. The contrast to countries without liver flukes is stark given that incidence of CCA is less than 3 per 100,000 elsewhere (USA, 1.67 ASR in 2014). To survive in hostile environs of the host biliary tract, the liver fluke excretes/secretes (ES) proteins and extracellular vesicles (EVs) for host-parasite communication, to manipulate the host responses, and to modify homeostasis, changes conducive to malignant transformation. This proposal targets components of liver fluke ES that drive the phenotypic hallmarks of cancer in the biliary tract: the growth mediator granulin, Ov-GRN-1 and extracellular vesicles (EVs) and their vesicle surface tetraspanins (TSPs). These mediators enter biliary epithelial cells, inducing proliferation, migration, angiogenesis, wound healing and proinflammatory cytokine IL- 6 production. We hypothesize that blocking internalization of Ov-GRN-1 and/or EVs into cholangiocytes will disrupt host-parasite communication, and in turn malignant transformation. We aim to test this hypothesis with the three specific aims. Aim 1. Assess the impact of using CRISPR-Cas9 to knock out liver fluke Ov-grn-1 and Ov-tsp genes on in vitro surrogates of pathogenicity and neoplasia. Aim 2. Characterize pathogenesis and cholangio-carcinogenicity of infection with gene edited (knockout) parasites in an informative rodent model of liver fluke infection and infection-induced bile duct cancer. Aim 3. Determine whether subunit Ov-GRN-1 and EV TSP vaccines protect against liver fluke infection and infection-induced cancer, and address mechanisms by which functional antibodies minimize pathology. We will utilize is a model of cholangiocarcinogenesis in which liver fluke infection is the confirmed risk factor. Innovations of the proposal include editing of the genome of the liver fluke using CRISPR-Cas9, targeting Ov-GRN-1 and EV surface tetraspanins with antibodies as an approach to anti-cancer therapy, and combining the findings of these innovations to develop a vaccine to block liver fluke infection. Fluke proteins that communicate at the host-parasite interface likely represent an Achilles' heel, and so targeting fluke-host communication in the form of an anti-fluke/ anti-cancer vaccine may ultimately defeat the disease. !